Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assist...Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assisted methods are important approaches for RNA direct detection,but its specificity will be limited when the fidelity of ligases is not ideal.The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection.Methods In this study,a dualcompetitive-padlock-probe(DCPLP)assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation.To verify the method,we employed dual competitive padlock probe-mediated rolling circle amplification(DCPLP-RCA)to genotype the CYP2C9 gene.Results The specificity was well improved through the competition and strand displacement of dual padlock probe,with an 83.26%reduction in nonspecific signal.By detecting synthetic RNA samples,the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L.Furthermore,clinical samples were applied to the method to evaluate its performance,and the genotyping results were consistent with those obtained using the qPCR method.Conclusion This study has successfully established a highly specific direct RNA SNP detection method,and provided a novel avenue for accurate identification of various types of RNAs.展开更多
单核苷酸多态性相关的拷贝数变异(copy number variant, CNV)在体外诊断和精准治疗中占据着重要地位,而其常用的荧光原位杂交技术(fluorescence in situ hybridization, FISH)无法区分具有单核苷酸差异的CNV。本研究利用交叉活性探针定...单核苷酸多态性相关的拷贝数变异(copy number variant, CNV)在体外诊断和精准治疗中占据着重要地位,而其常用的荧光原位杂交技术(fluorescence in situ hybridization, FISH)无法区分具有单核苷酸差异的CNV。本研究利用交叉活性探针定性与定量检测具有单核苷酸差异的拷贝数变异(copy number variant, CNV)。rs76711854所在以及下游9 514 bp长度片段通过PCR方法扩增获得,其基因型通过一代测序确认。此SNP的定性与定量分别通过探针法的定量PCR和数字PCR方法检测。rs76711854所在的CNV在针对G基因型探针出现分层,并在针对GA不同基因型的探针中均表现出A/G比例2:1。检测长度超过其下游9 kb以及更长的92 kb和203 kb的DNA片段,发现此CNV存在于前列腺癌细胞系DU145中,而子宫内膜癌细胞系中此203 kb范围拷贝数一致。本研究开发出以rs76711854位点的G/A片段为例,通过不同通道交叉定量检测的CNV方法,并推断DU145细胞的此处为多拷贝。展开更多
实现土壤失水过程的原位监测,对红壤区水土资源管理、自然灾害预警及防灾减灾具有重要意义。热脉冲法是目前土壤水热特性原位测量的常用方法,但红壤土失水时极易产生裂缝,单探针热脉冲法能否准确测定失水过程目前尚不清楚。通过室内模...实现土壤失水过程的原位监测,对红壤区水土资源管理、自然灾害预警及防灾减灾具有重要意义。热脉冲法是目前土壤水热特性原位测量的常用方法,但红壤土失水时极易产生裂缝,单探针热脉冲法能否准确测定失水过程目前尚不清楚。通过室内模拟土壤从饱和到干燥的连续失水过程,用时域反射法(TDR)作为标准对照,验证热脉冲方法测定土壤失水过程的准确性,并评估土壤裂缝产生后对单探针热脉冲法的影响。结果表明:随着土壤失水,土壤热导率K值由1.8 W/(m·K)减小到1.1 W/(m·K),热扩散率α值由1.0×10^(-8)m^(2)/s增加到3.0×10-8 m 2/s,比热容C值由1.8×108 J/(kg·K)减小到0.4×10^(8)J/(kg·K);接触热导率H值随着土壤失水具有台阶状变化特征,与土壤含水率变化速率的转折相吻合;与TDR测定结果相比,MAE和RMSE值分别为1.66和1.94 cm 3/cm^(3),R^(2)值为0.96;单探针热脉冲法能提供可靠的红壤土失水过程测定结果,且H值的变化能指征土壤裂隙的产生,可为土壤干湿循环过程、边坡和堤坝稳定性的动态监测提供技术和理论支撑。展开更多
单分子荧光原位杂交(single-molecule fluorescence in situ hybridization,smFISH)技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实...单分子荧光原位杂交(single-molecule fluorescence in situ hybridization,smFISH)技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实时研究。sm FISH适用于细胞、组织切片等多种类型生物样本。近年来,多种基于基础smFISH的改进技术被发明,进一步促进了该技术的实际应用。smFISH良好的RNA单分子可视化能力,使得其在发育生物学、神经生物学及肿瘤生物学等基础生物学科中得到了广泛的应用。本文综述了smFISH技术基本原理、smFISH技术的局限性、smFISH衍生技术方法、smFISH在不同生物学科中的应用进展,并对smFISH技术的发展前景做出展望。展开更多
文摘Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assisted methods are important approaches for RNA direct detection,but its specificity will be limited when the fidelity of ligases is not ideal.The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection.Methods In this study,a dualcompetitive-padlock-probe(DCPLP)assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation.To verify the method,we employed dual competitive padlock probe-mediated rolling circle amplification(DCPLP-RCA)to genotype the CYP2C9 gene.Results The specificity was well improved through the competition and strand displacement of dual padlock probe,with an 83.26%reduction in nonspecific signal.By detecting synthetic RNA samples,the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L.Furthermore,clinical samples were applied to the method to evaluate its performance,and the genotyping results were consistent with those obtained using the qPCR method.Conclusion This study has successfully established a highly specific direct RNA SNP detection method,and provided a novel avenue for accurate identification of various types of RNAs.
文摘实现土壤失水过程的原位监测,对红壤区水土资源管理、自然灾害预警及防灾减灾具有重要意义。热脉冲法是目前土壤水热特性原位测量的常用方法,但红壤土失水时极易产生裂缝,单探针热脉冲法能否准确测定失水过程目前尚不清楚。通过室内模拟土壤从饱和到干燥的连续失水过程,用时域反射法(TDR)作为标准对照,验证热脉冲方法测定土壤失水过程的准确性,并评估土壤裂缝产生后对单探针热脉冲法的影响。结果表明:随着土壤失水,土壤热导率K值由1.8 W/(m·K)减小到1.1 W/(m·K),热扩散率α值由1.0×10^(-8)m^(2)/s增加到3.0×10-8 m 2/s,比热容C值由1.8×108 J/(kg·K)减小到0.4×10^(8)J/(kg·K);接触热导率H值随着土壤失水具有台阶状变化特征,与土壤含水率变化速率的转折相吻合;与TDR测定结果相比,MAE和RMSE值分别为1.66和1.94 cm 3/cm^(3),R^(2)值为0.96;单探针热脉冲法能提供可靠的红壤土失水过程测定结果,且H值的变化能指征土壤裂隙的产生,可为土壤干湿循环过程、边坡和堤坝稳定性的动态监测提供技术和理论支撑。
